Autophagy consists of the sequestration of organelles and proteins in autophagic vesicles (AV) and degradation of this cargo through lysosomal fusion [1]. Autophagy allows tumor cells to survive metabolic and therapeutic stresses [2-5]. Multiple publications indicate therapy-induced autophagy is a key resistance mechanism to many anti-cancer agents. Chloroquine (CQ) (Compound 1, FIG. 1) derivatives block autophagy by inhibiting the lysosome [3,6,7]. Based on these findings, clinical trials combining cancer therapies with hydroxychloroquine (HCQ; FIG. 1 Compound 2), (which is safer than CQ to dose escalate) have been launched. Preliminary results indicate these combinations have activity [9-14], but it is still unclear if this activity is consistently due to the addition of HCQ. High micromolar concentrations of HCQ are required to inhibit autophagy.
While there is some pharmacodynamic evidence of autophagy inhibition with HCQ in cancer patients, it is inconsistent because adequate concentrations are not achieved in all patients. There is an unmet need to develop more potent inhibitors of autophagy. The design and synthesis of dimeric analogs of CQ, that exploit the thermodynamic advantages imparted by polyvalency [15,16], has been a subject of intensive study for over 10 years [17-19]. An early report by Vennerstrom[18] described the synthesis of heteroalkane-bridged bisquinolines as potential antimalarials, but none of the compounds had sufficient antimalarial activity to warrant further investigation. Subsequently, Sergheraert [17] reported that tetraquinolines, i.e., dimers of bisquinolines, afforded potent antimalarials, confirming the possibility that the application of the polyvalency strategy could afford increased potency, at least with respect to antimalarial activity. More recently, Lee[20] has described the potentiation of AKT inhibitors by fluorinated quinoline analogs. Solomon[21] has reported the preparation of “repositioned” chloroquine dimers, based on the use of a piperazine connector. These results suggest that these chloroquine analogs could serve as bases for the development of a new group of effective cancer chemotherapeutics.
We have examined the application of the strategy of polyvalency [15,16] to the synthesis of novel autophagy inhibitors by preparing a dimeric chloroquine from commercially available materials. We have recently reported a series of bis-4-aminoquinoline autophagy inhibitors (BAIs) that potently inhibit autophagy and impair tumor growth in vivo[22]. The structural motifs that are necessary for improved autophagy inhibition compared to CQ include the presence of two aminoquinoline rings and a triamine linker.
The multi-protein serine/threonine kinase mTORC1 (mammalian target of rapamycin complex 1) is a master regulator of catabolism and anabolism (Kim et al., 2002). For full activation, mTORC1 requires amino acid/Rag GTPase/Ragulator-dependent lysosomal localization to be in close proximity of Rheb (Ras homologue enriched in brain). The pentameric Ragulator protein complex (p18, p14, MP1, HBXIP, c7orf59) resides on the lysosomal surface and serves as a docking site for Rag GTPases when amino acids are present, which in turn directly interact with the raptor component of mTORC1, resulting in the lysosomal recruitment of mTORC1 (Bar-Peled et al., 2012; Sancak et al., 2010). Once on the lysosomal surface mTORC1 is fully activated by Rheb, which also resides on the cytoplasmic surface of the lysosome (Carroll et al., 2016). Rheb, the master activator of mTORC1, is negatively regulated by the tuberous sclerosis complex 1 (TSC1), TSC2 and TBC1D7 proteins. Environmental signals and intracellular conditions, including growth factor (GF) and amino acid (AA) availability, converge on TSC2, which in turn exerts its GTPase-activating protein (GAP) activity towards Rheb, shifting Rheb from its active GTP-bound state to its inactive GDP-bound conformation when GF/nutrient levels are low (Inoki et al., 2006). Therefore with the lysosomal residence of the Rag GTPases and Rheb, considered the two most proximal regulators of mTORC1, the lysosomal surface represents a critical signaling pivot where global cellular health information is integrated and translated into the activation status of mTORC1.
Lysosomal fusion and subsequent degradation of cargo-filled autophagic vesicles (AVs) allows for intracellular replenishment of nutrients, including AAs, sugars and nucleic acids (Jiang et al., 2015). Autophagy, an evolutionarily conserved homeostatic mechanism that allows cells to mitigate metabolic stresses of anabolism and catabolism, is directly regulated by mTORC1 via inhibitory phosphorylation of Unc-51-like kinase 1 (ULK1) at its serine-757 residue (Kim et al., 2011). When cellular health is compromised such as in the case of low AA levels, mTORC1 is inactivated due to its inability to be recruited by Rag GTPases to the lysosome, resulting in the initiation of autophagy, which recycles misfolded proteins into AA building blocks to restore cellular homeostasis. Aberrant autophagic-lysosomal activity and dysregulated mTORC1 signaling each have been demonstrated to allow tumor cells to resist therapeutic stresses of chemotherapy and targeted therapy, however attempts to clinically address these intertwined pro-tumorigenic mechanisms independently have had few durable responses with either PI3K/AKT/mTORC1-pathway targeted or autophagy-lysosome targeted monotherapy (Korfel et al., 2016; Wolpin et al., 2014). The combination of PI3K/AKT/mTORC1-pathway targeted agents with HCQ has been performed clinically with encouraging safety, tolerability and activity (Rangwala et al., 2014). However, pharmacokinetic (PK)-pharmacodynamic (PD) studies performed in patients receiving HCQ as cancer therapy have reported evidence of autophagy inhibition only in patients treated with the highest concentrations of HCQ that are inconsistently achieved in humans (Vogl et al., 2014). Therefore, there is an unmet need to develop more potent lysosomal inhibitors that can simultaneously influence autophagy and mTORC1 activity. Here we report the synthesis of a dimeric quinacrine (DQ) analog DQ661 that concurrently inhibits autophagy and mTORC1 by way of lysosome membrane permeabilization (LMP) and displacement of mTORC1 from the lysosomal compartment through disruption of Rag/Ragulator/lysosome interactions. This work identifies a novel pharmacological strategy to inhibit mTORC1 with in vivo activity in melanoma xenograft and pancreatic syngeneic mouse models.